Non-viral gene delivery system

ABSTRACT

The present invention concerns a composition comprising complexes of cationic chitosan oligomers derived from the cationic polysaccharide chitosan, wherein said cationic oligomers contain a weight fraction of less than 20% of oligomers with a Degree of Polymerization (DP)&lt;10 in addition to a weight fraction of less than 20% with DP&gt;50, and a nucleic acid. These compositions comprising well-defined cationic chitosan oligomers having a certain distribution of chain lengths, and nucleic acid are advantageous to achieve delivery of the nucleic acid into cells of a selected tissue, and to obtain in vivo expression of the desired molecules encoded for by the nucleic acid.

FIELD OF THE INVENTION

The present invention relates to a new non-viral delivery system fornucleic acids, and more specifically, to a system, which facilitates theintroduction of nucleic acid into cells in a host tissue afteradministration to that tissue. This system is based on a compositioncomprising chemically and physical-chemically well-defined cationicchitosan oligomers derived from biodegradable chitosan polysaccharidesthat efficiently delivers biologically active nucleic acids, such asoligo or polynucleotides that encodes a desired product, and facilitatesthe expression of a desired product in cells present in that tissue.

BACKGROUND ART

The concept of gene therapy is based on that nucleic acids, DNA, RNA canbe used as pharmaceutical products to cause in vivo production oftherapeutic proteins at appropriate sites. Delivery systems for nucleicacids are often classified as viral and non-viral delivery systems.Because of their highly evolved and specialized components, viralsystems are currently the most effective means of DNA delivery,achieving high efficiencies for both delivery and expression. However,there are safety concerns for viral delivery systems. The toxicity,immunogenicity, restricted targeting to specific cell types, limited DNAcarrying capacity, production and packaging problems, recombination anda very high production cost hamper their clinical use (Luo and Saltzman,2000). For these reasons, non-viral delivery systems have becomeincreasingly desirable in both basic research laboratories and clinicalsettings. However, from a pharmaceutical point of view, the way ofdelivery of nucleic acids still remains a challenge since a relativelylow expression is obtained in vivo with non-viral delivery systems ascompared to viral delivery systems (Saeki et al., 1997).

A variety of non-viral delivery systems, including cationic lipids,peptides or polymers in complex with plasmid DNA (pDNA), have beendescribed in the prior art (Boussif et al., 1995; Felgner et al., 1994;Hudde et al., 1999). The negatively charged nucleic acids interacts withthe cationic molecules mainly through ion-ion interactions, and undergoa transition from a free form to a compacted state. In this state thecationic molecules may provide protection against nuclease degradationand may also give the nucleic acid-cationic molecule complex surfaceproperties that favour their interaction with and uptake by the cells(Ledley, 1996).

Among these cationic molecules, the synthetic polymer polyethylenimine(PEI) have been shown to form stable complexes with pDNA and mediaterelatively high expression of the transgene both in vitro and in vivo(Boussif et al., 1995; Ferrari et al., 1997; Gautam et al., 2001). Forthis reason, PEI is often used as a reference system in the experimentalsetup. However, a rough correlation between toxicity and efficiency hasbeen suggested for PEI (Luo and Saltzman, 2000) and recent studies haveaddressed concerns about toxicity using PEI (Godbey et al., 2001; Putnamet al., 2001). Another drawback with PEI is that it is not biodegradableand it may therefore be stored in the body for a long time. Therefore,the search for effective and non-toxic biodegradable non-viral deliverysystems is highly desirable.

Most commonly, non-viral delivery systems have been delivered in vivo bythe parenteral route. After intravenous administration to mice,compacted nucleic acid-cationic molecule complexes deposited mainly inthe lung capillaries where the gene was expressed in the endothelium ofthe capillaries in the alveolar septi (Li and Huang, 1997; Li et al.,2000; Song et al., 1997) or even in the alveolar cells (Bragonzi et al.,2000; Griesenbach et al., 1998), but not in the epithelium. However,unformulated, naked DNA was rapidly degraded in the blood circulationbefore it reached its target and generally resulted in no geneexpression. In contrast, injection of naked DNA into skeletal muscleresulted in a dose-dependent gene expression (Wolff et al., 1990) whichwas further enhanced when complexed with a non-compacting but‘interactive’ polymer such as polyvinyl pyrrolidone (PVP) or polyvinylalcohol (PVA) (WO 9621470) (Mumper et al., 1996; Mumper et al., 1998).Thus, gene transfection in vivo is tissue-dependent in an unpredictableway and therefore remains a challenge.

Mucosal delivery of non-viral delivery systems has also been describedthat is delivery to the gastrointestinal tract, nose and respiratorytract (Koping-Hoggard et al., 2001; Roy et al., 1999), WO 01/41810. Withexception for the delivery to the nasal tissue where DNA in uncompactedform gives the best gene expression (WO 01/41810) compacted nucleicacid-cationic molecule complexes are preferred to uncompacted DNA when ahigh gene expression is required in a mucosal tissue.

In prior art, non-viral gene delivery systems are based on cationicpolymers such as chitosan of rather high molecular weight, often severalhundred kilodaltons (kDa) with 5 kDa as a lower limit, see for exampleMacLaughlin et al., 1998, Roy et al., 1999 and WO 97/42975. The majorreason is that polymers of lower molecular weight (<5 kDa) form unstablecomplexes with DNA, resulting in a low gene expression (Koping-Hoggard,2001). However, there are many drawbacks using cations of high molecularweight such as increased aggregation of compacted nucleic acid-cationicmolecule complexes and solubility problems (MacLaughlin et al., 1998).Further, there are several biological advantages of using cationicmolecules of lower molecular weights that is they generally show reducedtoxicity and reduced complement activation compared to cations of highermolecular weights (Fischer et al., 1999; Plank et al., 1999).

In the prior art some examples of the use of low molecular weightcations for complexation with nucleic acid have been described (Florea2001; Godbey et al., 1999; Koping-Hoggard, 2001; MacLaughlin, et al.,1998; Sato et al., 2001). However, these low molecular weight cationsform unstable compacts with DNA that separate in an electric field(agarose gel electrophoresis) resulting in no or a very low geneexpression in vitro, as compared to cations of higher molecular weights.This can be explained by that complexes formed between DNA and lowmolecular weight cations are generally unstable and dissociate easily(Koping-Hoggard, 2001). In fact, the dissociation of cationicmolecule-DNA compacts and release of naked DNA during agarose gelelectrophoresis has often been used as an assay to distinguishineffective formulations from effective ones in the literature (Fischeret al., 1999; Gebhart and Kabanov, 2001; Koping-Hoggard et al., 2001).Then, it is known from the prior art that complexes between DNA andcations should be stable to mediate a high gene expression.

The prior art contains various examples of methods for the delivery ofnucleic acids to the respiratory tract using non-viral vectors(Deshpande et al., 1998; Ferrari et al., 1997; Gautam et al., 2000). Werecently identified and characterized one such system based on theDNA-complexing polymer chitosan (Koping-Hoggard et al., 2001), a linearpolysaccharide, which can be derived from chitin. Chitosan-based genedelivery systems are also described in U.S. Pat. No. 5,972,707 (Roy etal., 1999), WO 98/01160 and in US patent application no.2001/0031497(Rolland et al., 2001).

Chitosan has been introduced as a tight junction-modifying agent forimproved drug delivery across epithelial barriers (Artursson et al.,1994). It is considered to be non-toxic after oral administration tohumans and has been approved as a food additive and also incorporatedinto a wound-healing product (Illum, 1998).

Chitosans comprise a family of water-soluble, linear polysaccharidesconsisting of (1→4)-linked 2-acetamido-2-deoxy-β-D-glucose (GlcNAc,A-unit) and 2-amino-2-deoxy-β-D-glucose, (GlcN, D-unit) in varyingcomposition and sequence (FIG. 1). The definition adopted here todistinguish between chitin and chitosan is based on the insolubility ofchitin in dilute acid solution and the solubility of chitosan in thesame dilute acid solution (Roberts, 1992).

The relative content of A- and D-units may be expressed as the fractionof A-units:

F_(A)=number of A-units/(number of A-units+number of D-units)

F_(A) is related to the percentage of de-N-acetylated units through therelation:

% de-N-acetylated units=100%·(1−F_(A))

Each D-unit contains a hydrophilic and protonizable amino group, whereaseach A-unit contains a hydrophobic acetyl group. The relative amounts ofthe two monomers (that is A/D=F_(A)/(1−F_(A))) can be varied over a widerange, and results in a broad variability in their chemical, physicaland biological properties. This includes the properties of the chitosansin solution, in the gel state and in the solid state, as well as theirinteractions with other molecules, cells and other biological andnon-biological matter.

The influence of the chemical structure of chitosans was recentlydemonstrated when chitosans were used in a non-viral gene deliverysystem (Koping-Hoggard et al., 2001). Chitosans of different chemicalcompositions displayed a structure dependent efficiency as gene deliverysystem. Only chitosans that formed stable complexes with pDNA gave asignificant transgene expression. Such complexes required that at least65% of the chitosan monomers were deacetylated.

Chitosans can be depolymerized either chemically or enzymatically toobtain chitosan polymers or oligomers of the desired molecular size.Various chemical degradation mechanisms can be used to depolymerizechitosans, that is acid hydrolysis, nitrous acid and oxidative-reductivedepolymerization. Ultrasonic depolymerisation of polymers mayalternatively be used, but these methods are very inconvenient forproducing very low molecular weights. Depolymerisation of chitosan bythe use of nitrous acid is a convenient way of preparing low-molecularweight chitosan, as described in for example U.S. Pat. No. 3,922,260 andU.S. Pat. No. 5,312,908. This mechanism involves deamination of aD-unit, forming 2,5-anhydro-D-mannose unit at the new reducing end,which can be reduced to 2,5-anhydro-D-mannitol using NaBH₄ as shown inFIG. 2. Alternatively, various enzymes can also be used to depolymerizechitosan, for instance U.S. 5,482,843, chitosanases, chitinases, andlysozyme. Also acid hydrolysis may be used to depolymerise chitosan(V{dot over (a)}rum et al., 2001, and references therein).

In the prior art, studies of the effect of molecular weight of chitosanon transfection efficiency in vitro of chitosan-pDNA complexes showed nosignificant dependence of the molecular weight in the size range 20-200kDa (Koping-Hoggard et al., 2001; MacLaughlin et al., 1998). However, inanother study (Sato et al., 2001) chitosans of 15 kDa and 52 kDa showedhigher gene expression than chitosan>100 kDa, while no gene expressionwas detected with a 1.3 kDa chitosan. Further, studies of geneexpression in vitro and in lung tissue in vivo using a series of lowmolecular weight chitosans (1.2 kDa, 2.4 kDa and 4.7 kDa) showed thatonly the 4.7 kDa chitosan mediated a significant gene expression(Koping-Hoggard, 2001).

Chitosans of different molecular weights have been used as components incomplexes for non-viral gene delivery. For example, US patentapplication no. 2001/0031497A refers to the use of small molecularweight chitosan as a component of the delivery system, that is chitosanin the range of 2-4 kDa Mw, which resulted in the smallest particle ofgene delivery system and also in an increased transfection of cells withthe condensed delivery system in vitro.

Chitosans of different molecular weights which are used in gene deliverysystems are normally unfractionated samples obtained from commercialsuppliers, and lower molecular weights are obtained from said samples bypartial degradation using degradation agents such as organic orinorganic acids, nitric acid or chitosan degrading enzymes. In allcases, the distribution of molecular weights remains relatively high. Asan example, a commercial chitosan with a weight average molecular weight(M_(w)) of 180.000 was analysed by size-exclusion chromatography using arefractive index detector and a multi-angle laser light scatteringdetector. FIG. 3A shows the elution profile, that is refractive indexdetector signal, which is proportional to the concentration of chitosan,combined with a plot of the calculated molecular weight (expressed aschitosan in the acetate salt form) as a function of the elution volume.It is evident that the sample contains molecular weights as high as 10⁶g/mol (1000 kDa) at the beginning of the peak and as low as 10⁴ (10 kDa)at the end of the peak. A recalculation of these data gives thecumulative molecular weight distribution (FIG. 3B). It may be inferredfrom these calculations that 12% (w/w) of the sample has a molecularweight below 40 kDa and 38% of the sample has a molecular weight below100 kDa. Likewise, 18% of the sample has a molecular weight above 300kDa and 9% has a molecular weight above 400 kDa. The sample is thuspolydisperse since it contains polymers of different molecular weightsor chain lenghts.

Chitosans may be supplied in the free amine form or as different saltssuch as chitosan chloride, chitosan glutamate and chitosan acetate. Thesalt-form influences the relationship between the molecular weight (M)and DP (the number of sugar residues per molecule). The followingequations describe this relationship between DP and M:

Free base: M=DP (161(1−F_(A))+203F_(A))=DP (161+42F_(A))

Chitosan chloride: M=DP (197.45(1−F_(A))+203F_(A))=DP (197.45+5.55F_(A))

Chitosan acetate: M=DP (221(1−F_(A))+203F_(A))=DP (221−18F_(A))

Chitosan glutamate: M=DP (308(1−F_(A))+203F_(A))=DP (308−105F_(A))

The weight average molecular weight (M_(w)) of a polydisperse sample maybe expressed as M_(w)=Σc_(i)M_(i)/Σc_(i) where c_(i) is theconcentration (g/l) of a particular molecular weight (M_(i)) within thedistribution) (Tanford, C. (1961) Physical chemistry of macromolecules,John Wiley and Sons, New York, Section 8b). Likewise, the number averagemolecular weight (M_(n)) may be expressed asM_(n)=Σc_(i)/Σ(c_(i)/M_(i)). In the case referred to above M_(w)=180 kDaand M_(n)=84.5 kDa, and the polydispersity index which is defined asM_(w)/M_(n) equals 2.1. A polydispersity near 2 is characteristic of alinear polymer which has been subjected to random depolymerisation(Tanford, C. (1961) Physical chemistry of macromolecules, John Wiley andSons, New York, Section 33a)

The distribution of chain lenghts following a random depolymerisation ofa linear polymer such as chitosan is given by the equation (Tanford(1961):W _(x) =xp ^(x−1)(1−p)²W_(x) is the weight fraction of chains containing x monomers (forchitosan the monomers are sugar residues) and p is the fraction ofintact linkages and 1−p is the fraction of cleaved lingages. The numberaverage degree of polymerisation (x_(n)) equals 1/(1−p). SinceM_(n)=M₀x_(n), where M₀ is the monomer equivalent weight, which is 203g/mol for a residue of N-acetyl-glucosamine when it occurs within achitosan chain and 161 g/mol for a residue of glucosamine in the freebase form when it occurs within a chitosan chain. For a given F_(A) theaverage M₀ becomes equal to 203·F_(A)+161·(1−F_(A)).

FIG. 4 shows SEC-MALLS chromatograms (4A), and differential (4B) andcumulative (4C) molecular weight distributions of a chitosan, which hasbeen depolymerised by nitrous acid to obtain different weight averagemolecular weights in the range from 41.500 to 13.400. It is clearlyshown that the calculated molecular weight distributions remain broad.These data clearly demonstrate that chitosans of different molecularweights which are produced from a high molecular weight by partialdegradation remain polydiserse and contain chains of widely differingmolecular weights.

The molecular weight distribution of a polymer may be modified byselectively removing certain parts of the distribution. Chitosan sampleswith relatively short chains may be fractionated by gel filtration toobtain individual oligomers or fractions with relatively narrowmolecular weight distributions. One example is given by Tøommeraas etal. (2001) who obtained purified chitosan oligomers in the range of 2-10residues per chain.

Samples with higher molecular weights may also be fractionated by gelfiltration as demonstrated for chitosans by Ottøy et al. (1996).Typically, fractions with M_(w)/M_(n) values of 1.2-1.5 was obtained byfractionating a normally polydisperse sample with M_(w)=270.000 using agel filtration column containing Sepharose CL-4B and Sepharose CL-6B.

In an alternative method polydisperse chitosans may be fractionated bydialysis or membrane techniques which allow selective removal of theshortest chains, and where the resulting distribution depends on theinitial distribution as well as the membrane characteristics porosityand transport coefficients and the operating conditions.

According to the present invention it was surprisingly discovered thatchitosans of a single chain lenght or chitosans with narrow molecularweight distributions had different properties as complexing agents ingene delivery than other samples of comparable M_(w) or M_(n), but withbroader molecular weight distributions.

Another disadvantage of many cations used for complexation of nucleicacid e.g. PEI, polylysine and chitosan is that they are roughlyprocessed bulk chemicals with a broad molecular weight distribution andhence rather undefined (Godbey et al., 1999). It is well establishedthat such chemicals may display a batch to batch variation. Therefore,from a pharmaceutical point of view, well-defined polycations having anarrow molecular weight distribution are preferred.

Another disadvantage using broad molecular weight polycations forcomplexation of nucleic acids and subsequent transfection is that chainsof differents lenghts may have different complexation and transfectioneffectivities.

SUMMARY OF THE INVENTION

The present invention is concerned with a composition comprisingcomplexes of:

-   -   (a) cationic chitosan oligomers derived from the cationic        polysaccharide chitosan wherein said cationic oligomers contain        a weight fraction of less than 20% of oligomers with a Degree of        Polymerization (DP)<10 in addition to a weight fraction of less        than 20% with DP>50; and    -   (b) a nucleic acid.

According to the present invention it has unexpectedly been found thatcompositions comprising well-defined cationic chitosan oligomers havinga certain distribution of chain lengths, and nucleic acid areadvantageous to achieve delivery of the nucleic acid into cells of aselected tissue and to obtain in vivo expression of the desiredmolecules encoded for by the nucleic acid.

It is another object of the invention to provide a method of preparingcompositions according to the invention, comprising the steps of:

-   -   (a) exposing said cationic chitosan oligomers to an aqueous        solvent,    -   (b) mixing the aqueous solution of step (a) with said nucleic        acid in an aqueous solvent, and    -   (c) reduce the volume of the product solution obtained in        step (b) to achieve a desired concentration of the said        composition.

It is yet another object of the present invention to provide a method ofadministering a nucleic acid to a mammal, by introduction of thecomposition, of the invention, into the mammal.

A further object of the present invention are the use of the compositionof the invention in the manufacture of a medicament for phrophylactic ortherapeutic treatment of a mammal, or in the manufacture of a diagnosticagent for the use in in vitro or in vivo diagnostic methods.

These and other objects of the invention are provided by one or more ofthe embodiments described below.

DETAILED DESCRIPTION OF THE INVENTION

The composition according to the present invention can be derived fromcationic polysaccharide chitosan by the use of chemical or enzymaticmethods.

A preferred composition of the invention is wherein said cationicoligomers contain preferably a weight fraction of less than 20% ofoligomers with DP<12 in addition to a weight fraction of less than 20%with a DP>40 and most preferably a weight fraction of less than 20% ofoligomers with DP<15 in addition to a weight fraction of less than 20%with a DP>30.

Compositions comprising complexes between low molecular weight cationicchitosan oligomers and nucleic acid are described, wherein the cationicchitosan oligomers have well-defined chain lengths, narrow distributionof chain lengths and a well-defined chemical composition. Typically, thecationic chitosan oligomer has a molecular weight between 500 and 10,000Da, preferably between 1,200 and 5,000 Da and most preferably between3,000 and 4,700 Da. Typically the cationic chitosan oligomer has afraction of A-units (F_(A)) of 0-0.35 (65-100% de-N-acetylated units),preferably between 0-0.1 (90-100% de-N-acetylated units) and mostpreferably between 0-0.01 (99-100% de-N-acetylated units). Suitably,said nucleic acid comprises a coding sequence that will express itsfunction when said nucleic acid is introduced into a host cell.

According to one embodiment of the invention, said oligomers are derivedfrom cationic polysaccharide chitosans followed by fractionating apolydisperse oligomer pool into oligomers having well-defined chainlengths, narrow distribution of chain lengths and a fraction of A-units(F_(A)) of 0-0.35 (65-100% de-N-acetylated units), preferably between0-0.1 (90-100% de-N-acetylated units) and most preferably between 0-0.01(99-100% de-N-acetylated units). Typically, said oligomers consist of6-50 monomer units, preferably of 10-30 monomer units and mostpreferably of 15-25 monomer units, having a molecular weight between3,000 and 4,700 Da, and a F_(A) of less than 0.01 (more than 99%de-N-acetylated units).

According to another embodiment of the composition of the invention,said nucleic acid is selected from the group consisting of RNA and DNAmolecules. These RNA and DNA molecules can be comprised of circularmolecules, linear molecules or a mixture of both. Preferably, saidnucleic acid is comprised of plasmid DNA.

According to a preferred embodiment of the present invention, saidnucleic acid comprises a coding sequence that will express its functionwhen said nucleic acid is introduced into a host cell. For instance itcan encode a biologically active product, such as a protein, polypeptideor a peptide having therapeutic, diagnostic, immunogenic, or antigenicactivity.

The present invention is also concerned with compositions as describedabove wherein said nucleic acid comprises a coding sequence encoding aprotein, an enzyme, a polypeptide antigen or a polypeptide hormone orwherein said nucleic acid comprises a nucleotide sequence that functionsas an antisense molecule, such as RNA.

Preferably the composition of the invention has a pH range between 3.5and 8.

The composition of the invention can also preferably be derivatized withtargeting ligands and/or stabilizing agents.

A further aspect of the invention is related to the liquid droplet sizeof said composition after nebulization. Preferably, the droplet size ofthe composition of the invention is essentially equal to the dropletsize of naked pDNA after nebulization.

The present invention is also directed to a method for preparing thepresent composition, said method comprising the steps of: providing thepresent cationic chitosan oligomer as described above, (a) exposing saidcationic chitosan oligomers to an aqueous solvent in the pH range3.5-8.0, (b) mixing the aqueous solution of step (a) with said nucleicacid in an aqueous solvent and (c) dehydrating the product solutionobtained in step (b) to achieve a high concentration of the compositionbefore administration in vivo. Step (c) can be obtained by (1)evaporating the liquid of the product solution in step (b) to obtain thedesired concentration, or (2) lyophilize the product solution in step(b) followed by reconstitution of the lyophilizate to obtain the desiredconcentration of the composition. Typically, said nucleic acid ispresent at a concentration of 1 ng/ml-300 μg/ml, preferably 1 μg/ml-100μg/ml and most preferably 10-50 μg/ml in step (b) and 10 ng/ml-3,000μg/ml, preferably 10 μg/ml-1,000 μg/ml and most preferably 100-500 μg/mlin step (c) using the evaporating method (1).

It should be understood, that a person skilled in the art can form thepresent composition at different amine/phosphate charge ratios toinclude negative, neutral or positive charge ratios. However a preferredembodiment is wherein the composition of the invention has a netpositive charge.

The present invention is further concerned with a method ofadministering nucleic acid to a mammal, using the composition of thepresent invention, and introducing the composition into the mammal.Preferably, said composition is introduced into the mammal byadministration to mucosal tissues by pulmonary, nasal, oral, buccal,sublingual, rectal, or vaginal routes. According to another preferredembodiment, said composition is introduced into the mammal by parenteraladministration such as intravenous, intramuscular, intradermal,subcutaneous or intracardiac administration.

The present invention is also concerned with use of the composition ofthe invention in the manufacture of a medicament for prophylactic ortherapeutic treatment of a mammal, or in the manufacture of a diagnosticagent for the use in in vivo or in vitro diagnostic methods, andspecifically in the manufacture of a medicament for use in gene therapy,antisense therapy or genetic vaccination for prophylactic or therapeutictreatment of malignancies, autoimmune diseases, inherited disorders,pathogenic infections and other pathological diseases.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by the way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical composition of chitosan, where a fragment ofthe chitosan chain contains one residue of N-acetyl-β-D-glucosamine(A-unit) and 3 residues of β-D-glucosamine (D-units). The amino group ofthe D-units may be on a protonated or unprotonated form depending on pH.

FIG. 2 a shows the chemical structure which is obtained afterdepolymerisation of a chitosan by acid or by a chitosanase. Acids cleavepreferentially the glycosidic bond following an A-unit (A-unit at thenewly formed reducing end). Enzymes vary in their specificities byhydrolysing both kinds of residues.

FIG. 2B shows the depolymerisation of chitosan by nitric acid, whichonly attacks D-residues, which are rearranged to form2,5-anhydro-D-mannose.

FIG. 3 shows results where a commercial chitosan with a weight averagemolecular weight (M_(w)) of 180.000 was analysed by size-exclusionchromatography using a refractive index detector and a multi-angle laserlight scattering detector. Columns: TSK G6000PWXL, 5000PWXL and 4000PWXL (serially connected). Eluent: 0.2 M ammonium acetate, pH 4.5. RIdetector: Optomed DSP (Wyatt). Light scattering detector: DAWN DSP(Wyatt). Processing parameters (Astra software v. 4.70.07): dn/dc=0.142ml/g (determined off-line for chitosan acetate, the value was found tobe independent of F_(A)). A₂: 5.0·10⁻³ mol·ml·g⁻².

3A: Elution profile, that is refractive index detector signal, which isproportional to the concentration of chitosan, combined with a plot ofthe calculated molecular weight in this case expressed as chitosan inthe acetate salt form as a function of the elution volume.

3B: The cumulative molecular weight distribution calculated from thedata given in 3A.

FIG. 4 shows SEC-MALLS chromatograms (4A), and differential (4B) andcumulative (4C) molecular weight distributions of a chitosan, which hasbeen depolymerised by nitrous acid to obtain different weight averagemolecular weights in the range from 41.500 to 13.400. Experimentalconditions were the same as in FIG. 3.

FIG. 5: Calculated cumulative (A) and differential (B) molecular weightdistributions corresponding to the Kuhn distribution for chitosandepolymerised to obtain 100, 50, 20 and 10 residues (DP_(n)).

FIG. 6: Size-exclusion chromatograms of a fully de-N-acetylated chitosan(F_(A)<0.001) which has been depolymerized by a) nitrous acid andreduced with NABH₄ (N1-N4) or b) chitosanase (E1-E4) (Superdex 30; two2.5×100 cm columns in series, eluent: 0.15M ammonium acetate, pH 4.5,flow rate 0.8 m/min). DP=6 indicates the elution volume of a fullyde-N-acetylated chitosan hexamer.

FIG. 7: SEC-MALLS chromatograms (7A) of a fully de-N-acetylated chitosan(F_(A)<0.001), which has been depolymerised by nitrous acid and reducedwith NaBH₄ (un-fractionated sample) and fractions N1-N4 obtained asdescribed in FIG. 6. The experimental conditions were the same as inFIG. 3 except that a single column (TSK G3000 PWXL) was used. FIG. 7Bshows the corresponding cumulative molecular weight distributionscalculated from the data given in 7A.

FIG. 8: SEC-MALLS chromatograms (8A) of a chitosan, which has beendepolymerised by a chitosanase (un-fractionated sample) and fractionsE1-E4 obtained as described in FIG. 6. The experimental conditions werethe same as in FIG. 3 except that a single column (TSK G3000 PWXL) wasused. FIG. 8B shows the corresponding cumulative molecular weightdistributions calculated from the data given in 7A.

FIG. 9 shows in vivo lung luciferase expression (pg/mg) 3 days afterintra-tracheal administration of 25 μg pLuc in mice (four animals pergroup). Complexes between chitosan oligomers and pLuc were prepared atan amine/phosphate charge ratio of 60:1 (+/−). The significantly highestluciferase expression was obtained with pLuc complexed with the chitosanoligomer N0 having 18 as the number average degree of polymerisation, asdetermined by ¹³C-NMR-spectroscopy. Statistical differences between meanvalues were investigated using ANOVA. Differences between group meanswere considered significant at P<0.05.

FIG. 10 shows in vivo lung luciferase expression (pg/mg) 3 days afterintra-tracheal administration of 25 μg pLuc in mice (four animals pergroup). The chitosan oligomer N0 having 18 as the number average degreeof polymerization was fractionated into four different samples havingwell-defined and narrow distributions of their degrees ofpolymerization. Complexes between chitosan oligomers and pLuc wereprepared at an amine/phosphate charge ratio of 60:1 (+/−). Complexesbased on the fraction containing oligomers having chain lenghts between15-21 monomer units (N3), showed significantly (p<0.05) higher geneexpression compared to complexes based on the unfractionated sample N0having 18 as the number average degree of polymerization. Statisticaldifferences between mean values were investigated using ANOVA.Differences between group means were considered significant at P<0.05.

FIG. 11 shows results of the agarose gel retardation assay. Complexesbetween chitosan oligomers and pLuc were prepared at an amine/phosphatecharge ratio of 60:1 (+/−). With increasing molecular weight (degree ofpolymerization) of the chitosan oligomer, a higher stability of formedcomplexes was observed. Thus, complete retention of pLuc was detectedwith complexes formed with the fraction containing 36-50 monomer units(N1) as compared to complexes formed with 15-21 monomer units (N3).

FIG. 12 shows the luciferase gene expression in vitro after incubating293 cells with two batches of fractionated low molecular weight cationicchitosan oligomers (N1 and E1) prepared 9 months apart and commercialchitosan (Protasan UPG 210) ordered 3 years apart, respectively. Thegene expression varied 10-fold between the two batches of Protasan UPG210 complexed with pLuc at an amine/phosphate charge ratio of 2.4:1(+/−) but not significantly between the two batches of fractionated lowmolecular weight cationic chitosan oligomers (N1 and E1) complexed withpLuc at an amine/phosphate charge ratio of 10:1 (+/−). Statisticaldifferences between mean values were investigated using ANOVA.Differences between group means were considered significant at P<0.05.

FIG. 13 shows the liquid droplet size (mass median diameter, MMD) afteraerosolization of compositions containing cations complexed with pLuc.Fractions of chitosan oligomers containing 15-21 (N3) and 36-50 (N1)monomer units and an ultra pure chitosan, Protasan UPG 210 (UPC), werecomplexed with pLuc at an amine/phosphate charge ratio of 60:1 (+/−) and3:1 (+/−), respectively. The MMD was clearly dependent on thecomposition. The smallest droplet size was obtained with naked pLuc andthe composition containing 15-21 monomer units (N3) complexed with pLuc.Statistical differences between mean values were investigated usingANOVA. Differences between group means were considered significant atP<0.05.

Using the expression of a reporter protein, luciferase, as a model for atherapeutic protein in an in vivo lung model, it was found thatformulations comprising plasmid DNA and a certain composition ofchitosan oligomers having well-defined chain lenghts, distribution ofchain lenghts, and chemical composition, are advantageous to achievedelivery of the nucleic acid into cells of a selected tissue and toobtain in vivo expression of the desired molecules encoded for by thenucleic acids.

It was found that a chitosan oligomer fraction, prepared from chitosan,having a number-average degree of polymerization of 18 (DP_(n)=18, asdetermined by ¹³C-NMR-spectroscopy), showing a relatively narrow sizedistribution as compared to the Kuhn-distribution and having more than99% D-units (F_(A)<0.01), formed stable complexes (as revealed byagarose gel electrophoresis) with pLuc at an amine/phosphate chargeratio of 60:1 (+/−). A significantly higher in vivo lung luciferase geneexpression was obtained with the polydisperse DP_(n)=18 sample comparedto monodisperse chitosan oligomers having 6, 10 and 12 monomer unitsthat formed unstable complexes with pLuc at an amine/phophate chargeratio of 60:1 (+/−). The fact that stable complexes resulted in a highergene expression than unstable complexes is in agreement with the priorart (Fischer et al., 1999; Gebhart and Kabanov, 2001; Koping-Hoggard etal., 2001). However, a decrease in luciferase expression was detectedwith stable complexes formed with chitosan oligomers having higheraverage molecular sizes than the DP_(n)18 sample. The fraction DP_(n)18was further fractionated into fractions having more narrow distributionsthat is 10-14 monomer units (N4), 15-21 monomer units (N3), 22-35monomer units (N2) and 36-50 monomer units (N1). Complexes between thefraction having 15-21 monomer units and pLuc resulted unexpectedly inthe highest in vivo lung gene expression although unstable complexeswere formed at an amine/phosphate charge ratio of 60:1 (+/−). Thefraction 10-14 monomer units also formed unstable complexes with pLucand resulted only in a modest luciferase expression.

Also, aerosolisation of complexes between the fraction having 15-21monomer units and pLuc resulted in comparable droplet sizes as anaerosolised solution of naked pLuc. In contrast, aerosolisation of thefraction having 36-50 monomer units complexed with pLuc and UPC(approximately 1000-mer) complexed with pLuc resulted in a 2 and 3-foldhigher droplet size, respectively. This formulation-dependent effect onthe droplet size might be explained by an increased viscosity of thesolution with increasing molecular weight of the cation, thus producingdroplets of a larger size.

EXAMPLES Example 1 Preparation of Low-Molecular Weight Chitosans

Chitosan Protasan UP G 210 (F_(A)=0.17, weight-average molecular weightof 162,000) was obtained from Pronova Biomedical AS, Oslo, Norway. Thelow-molecular weight oligomer of N-glucosamine was obtained by chemicaldepolymerisation of chitosan using NaNO₂ and subsequent reduction byNaBH₄ as described by Tømmeraas et al., 2001, where the molecular weightwas controlled by the amount of NaNO₂ relative to the amount ofchitosan. The fraction of acetylated units was controlled byheterogeneous deacetylation to obtain F_(A) of less than 0.001 asdetermined by proton NMR-spectroscopy as described previously (V{dotover (a)}rum et al., 1991). Typically, 1.0 gram of chitosan wasdissolved in 100 ml of 2.5% aqueous acetic acid, dissolved oxygen wasremoved by bubbling nitrogen gas through the solution for 5 minutes, and5 ml of a freshly prepared solution of NaNO₂ in distilled water (10mg/ml) was added. The reaction was allowed to proceed for 4 hours indarkness, whereafter the depolymerized chitosan was conventionallyreduced by adding 3 grams of NaBH₄ overnight in darkness. The pH wasadjusted to 4.5 using acetic acid. The solution was dialysed (Medicelldialysis tubing, MWCO 12000-14000) three times against 0.2M NaCl and sixtimes against distilled water and lyophilized, to obtain thelow-molecular weight oligomer as their hydrochloride salt.Alternatively, the low-molecular weight oligomers were obtained byenzymatic depolymerisation using a chitosanase from Streptomyceusgriseus (Sigma C 9830 or Sigma C 0794) where the molecular weight iscontrolled by the amount of enzyme relative to the amount of chitosanand the incubation time. 0.5 gram of chitosan was dissolved at aconcentration of 20 mg/ml in 0.1M sodium-acetate/acetic acid buffer (pH5.5) and 0.65 units of Chitosanase (Sigma C 0794) was added to thechitosan solution and incubated for 18 hours at 37° C. The enzymereaction was stopped by decreasing the pH to 2 and then boiled for 5minutes. The depolymerized chitosan was dialysed and lyophilized asdescribed above, to obtain the low molecular weight enzyme-degradedchitosan as their hydrochloride salts.

Example 2 Preparation and Characterization of Fractionated Samples

The low-molecular weight chitosans prepared as described in Example 1were fractionated by size-exclusion chromatography on two 2.5×100 cmcolumns connected in series as described previously (Tømmeraas et al.,2001). Fractions of 4 mL were collected and pooled according to thechromatograms shown in FIG. 6 (a and b), to obtain 4 fractions differingin molecular weight designated

N1 (nitrous acid degraded) or E1 (chitosanase degraded)

N2 (nitrous acid degraded) or E2 (chitosanase degraded)

N3 (nitrous acid degraded) or E3 (chitosanase degraded)

N4 (nitrous acid degraded) or E4 (chitosanase degraded) TABLE 1 Thesamples were analyzed by SEC-MALLS, which yielded the following chainlength distributions (average of 3 injections): Sample DP_(w) DP_(n)DP_(w)/DP_(n) Unfractionated N0 31 25 1.22 (nitrous acid degraded) N1 4440 1.09 N2 27 26 1.03 N3 20 19 1.03 N4 14 13 1.04 Unfractionated E0 2721 1.31 (chitosanase degraded) E1 50 44 1.12 E2 33 30 1.06 E3 25 23 1.03E4 17 16 1.07wherein DPw = weight average DPand DPn = number average DP

Example 3 Formulation and in vivo Lung Gene Expression

A polydisperse cationic chitosan oligomer fraction having a degree ofpolymerization (DP) between 6-50 (number-average DP of 18 as determinedfrom the non-reducing ends in the ¹³C-nmr-spectrum, N0) and well-definedcationic oligomers, having DP's of 6, 10, 12, 10-14 (N4), 15-21 (N3),22-35 (N2), 36-50 (N1) were prepared from chitosan according to themethods described in Example 1 and Example 2. Firefly luciferase plasmidDNA (pLuc) was purchased from Aldevron, Fargo, N.D., USA. Stocksolutions of cationic chitosan oligomers (2 mg/ml) were prepared insterile distilled deionized water, pH 6.2±0.1 followed by sterilefiltration. Complexes between cationic chitosan oligomers and pLuc wereformulated at a charge ratio of 60:1 (+/−) by adding cationic oligomerand then pLuc to sterile water under intense stirring on a vortex mixer(Heidolph REAX 2000, KEBO Lab, Sp{dot over (a)}nga, Sweden). After 15min the complexes were concentrated by mild evaporation under vacuum ina SpeedVac Plus centrifuge (Savant Instruments, Holbrook, N.Y.) forapproximately 90 min to obtain pLuc concentrations of around 250 μg/ml)(Koping-Hoggard et al., 2001). In addition, pLuc was formulated with PEI25 kDa (Aldrich Sweden, Stockholm, Sweden) and an ultra pure chitosan,Protasan UPG 210 (Pronova Biopolymer, Oslo, Norway) at previouslyoptimized conditions, charge ratio 5:1 (+/−) and 3:1 (+/−) respectively(Bragonzi et al., 2000; Koping-Hoggard et al., 2001).

Mice (male Balb/c, 6-8 weeks old, 4 animals per group, Charles River,Uppsala, Sweden) were anesthesized with ketamin/xylazine (5/20 vol %,0.1 ml/10 g of body weight), and the trachea was surgically exposed witha 0.5 cm long skin incision in the neck. 100 μl of the complexesdescribed aboved was slowly administrated dropvise into the trachea andthe mice were sutured. At 72 h after administration, the animals weresacrified by carbon dioxide and the lungs were surgically removed,washed in PBS and 0.3 ml ice-cold luciferase lysis buffer (Promega,Madison, Wis.) with a protease inhibitor coctail (Complete, BoehringerMannheim Scandinavia AB, Bromma, Sweden) was added. The tissue sampleswere quickly frozen in liquid nitrogen and stored at −80° C. untilanalysis.

In a cold room, the tissue samples were homogenized in a bead beater(Biospec Products, Inc., OK) followed by centrifugation (Centrifuge5403, Eppendorf-Nethelar-Hinze GmbH, Hamburg, Germany) at 4° C. and15,000 rpm for 10 min. An amount of 50 μl of the clear supernantant fromeach test tube was mixed with 50 μl of luciferase reagent (Promega) andanalyzed by a luminometer (Mediators PhL, Vienna, Austria) with anintegration time of 8 s. In order to quantify the luciferase expression,a standard curve of luciferase (Sigma, St. Louise, Mo.) was prepared byadding defined amounts of the luciferase standard to the supernatants ofhomogenized tissues from untreated control animals. The total proteincontent in each sample was analyzed by the BCA assay (Pierce, Rockford,Ill.) and quantified using BSA (bovine serum albumin) as a referenceprotein. The absorbance was measured at 540 nm on a microplate reader(Multiscan MCC/340, Labsystems Oy, Helsinki, Finland).

Results of the gene transfection efficiency in mouse lungs 72 h afteradministration of pLuc complexed with cationic chitosan oligomers ofvarious degree of polymerization (molecular weight) are shown in FIG. 9.Surprisingly, the significantly highest luciferase expression wasobtained with pLuc complexed with a chitosan oligomer N0 having 18 asthe number average degree of polymerization.

The results of the gene transfection efficiency in mouse lungs 72 hafter administration of pLuc complexed with cationic chitosan oligomersof various degree of polymerization (molecular weight) are shown in FIG.10. The chitosan oligomer N0 having 18 as the number average degree ofpolymerization was fractionated, as described in Example 2, into foursamples having well-defined and narrow distributions of their degrees ofpolymerization. Surprisingly, the fraction containing chitosan oligomershaving chain lenghts between 15-21 monomer units (N3), showed highergene expression than PEI and significantly higher gene expressioncompared to the un-fractionated sample N0 having 18 as the numberaverage degree of polymerization. The results of the agarose gelretardation assay are shown in FIG. 11. With increasing molecular weight(degree of polymerization) of the chitosan oligomer, the stability offormed complexes increases. Thus, almost complete retention of pLuc wasdetected with complexes formed with the fractions containing 22-35 (N2)and 36-50 (N1) monomer units as compared to complexes formed with 10-14(N4) and 15-21 (N3) monomer units. The unfractionated sample N0 having18 as the number average degree of polymerization also formed stablecomplexes with pDNA. A higher in vivo gene expression (FIG. 10) wassurprisingly obtained with the less stable 15-21 (N3) complexes comparedto the stable complexes formed with DPn18 (N0).

Example 4 In vitro Gene Expression

Two different batches of fractionated low molecular weight cationicchitosan oligomers; N1 and E1, as described in example 2 and prepared 9months apart, and commercial chitosan (Protasan UPG 210, batch 1:apparent viscosity of 70 mPas, batch 2: apparent viscosity of 146 mPas)ordered 3 years apart were complexed with pLuc at charge ratios of 10:1(+/−) and 2.4:1 (+/−), respectively, as described in Example 2. StablepDNA complexes were used.

24 h before transfection, the epithelial human embryonic kidney cellline 293 (ATCC, Rockville, Md., USA) were seeded at 70% confluence in96-well tissue culture plates (Costar, Cambridge, UK). Prior totransfection, the cells were washed and then 50 μl (corresponding to0.33 μg pLuc) of the polyplex formulations was added per well. After 5 hincubation, the formulations were removed and 0.2 ml of fresh culturemedium was added. The medium was changed every second day forexperiments exceeding two days. At 96 h and 144 h, cells were washedwith PBS (pH 7.4), lysed (Promega) and luciferase gene expression wasmeasured with a luminometer (Mediators PhL). The amount of luciferaseexpressed was determined from a standard curve prepared with fireflyluciferase (Sigma) and total cell protein was determined using thebichinchoninic acid test (Pierce).

The results of the luciferase gene expression in vitro after incubating293 cells with two batches of fractionated low molecular weight cationicchitosan oligomers; N1 and E1 and commercial chitosan Protasan UPG 210,respectively, are shown in FIG. 12. The gene expression varied 10-foldbetween the two batches of Protasan UPG 210 but not significantlybetween the two batches of the fractionated low molecular weightcationic chitosan oligomers N1 and E1.

Example 5 Droplet Size after Aerosilisation

Complexes between cationic chitosan oligomers and pLuc were prepared asdescribed in Example 3 to obtain pLuc concentrations of 500 μg/ml. As acontrol, an ultra pure chitosan (UPC, degree of polymerization around1000) complexed with pLuc were used at optimal conditions, charge ratio3:1 (+/−) (Koping-Hoggard et al., 2001). Aerosols containing complexesbetween cationic chitosan oligomers and pLuc were produced with the useof a nebulization catheter (Trudell Medical International, LondonOntario, Canada) containing liquid- and gas (air)-channels. Firstly, 100μl of the complex solution was loaded into a liquid reservoir coupled tothe nebulization catheter (liquid inlet). Then, to obtain aerosols,pulses of pressurized air (3.5 bar) was applied for short time periodsover the liquid reservoir (20 ms) and the gas channels of thenebulization catheter (50 ms). The droplet size of produced aerosols wasmeasured with a Mastersizer X (Malvern instruments Ltd., Malvern, UK).

The liquid droplet size (mass median diameter, MMD) after aerosolisationof compositions containing cations complexed with pLuc are shown in FIG.13. The MMD was clearly dependent on the composition. The smallestdroplet size was obtained with “naked” pLuc and the compositioncontaining 15-21 monomer units (N3) complexed with pLuc.

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1. A composition comprising complexes of: (a) cationic chitosanoligomers derived from the cationic polysaccharide chitosan wherein saidcationic oligomers contain a weight fraction of less than 20% ofoligomers with a Degree of Polymerization (DP)<10 in addition to aweight fraction of less than 20% with DP>50, wherein the fraction ofN-acetylated units (F_(A))of said chitosan oligomers is 0-0.1; and (b) anucleic acid.
 2. The composition of claim 1, wherein said cationicchitosan oligomers are obtained from chitosan using chemical orenzymatic methods.
 3. The composition of claim 1, wherein said cationicoligomers contain a weight fraction of less than 20% of oligomers withDP<12 and a weight fraction of less than 20% with a DP>40.
 4. (canceled)5. The composition of claim 1, wherein said composition essentially hasa net positive charge ratio.
 6. The composition of claim 1 wherein saidchitosan oligomers are derivatized with targeting ligands andstabilizing agents.
 7. The composition of claim 1, wherein saidcomplexes comprise a coding sequence that will express its function whensaid nucleic acid is introduced into a host cell.
 8. The composition ofclaim 7, wherein said nucleic acid is selected from the group consistingof DNA and RNA molecules.
 9. The composition of claim 8, wherein saidcomposition has a pH in the range of 3.5 to
 8. 10. The composition ofclaim 9, wherein said composition after aerosolisation essentially has acomparable droplet size as to a composition consisting of only nucleicacid at equal concentrations of nucleic acid.
 11. A method of preparingthe composition of claim 1, comprising the steps of: (a) exposing saidcationic chitosan oligomer to an aqueous solvent; (b) mixing the aqueoussolution of step (a) with said nucleic acid in an aqueous solvent; and(c) reducing the volume of the product solution obtained in step (b) toachieve a desired concentration of the said composition.
 12. A method ofadministering nucleic acid to a mammal, using the composition of claim1, and introducing the composition into the mammal.
 13. The method ofclaim 12, wherein the composition is introduced into the mammal byadministration to mucosal tissues by pulmonary, nasal, oral, buccal,sublingual, rectal or vaginal routes.
 14. The method of claim 12,wherein the composition is introduced into the mammal by administrationto submucosal tissues by parenteral routes that is intravenous,intramuscular, intradermal, subcutaneous or intracardiac administration,or to internal organs, blood vessels or other body surfaces or cavitiesexposed during surgery.
 15. A method of claim 12 comprising thecomposition of claim 1, whereby said nucleic acid is capable ofexpressing its function inside said cell.
 16. The use of the compositionof claim 1, in the manufacture of a medicament for prophylactic ortherapeutic treatment of a mammal, or in the manufacture of a diagnosticagent for use in in vivo or in vitro diagnostic methods.
 17. The use ofthe composition of claim 16 in the manufacture of a medicament for usein gene therapy, antisense therapy, or genetic vaccination forprophylactic or therapeutic treatment of malignancies, autoimmunediseases, inherited disorders, pathogenic infections and otherpathological diseases.
 18. The composition of claim 3, wherein saidcationic oligomers contain a weight fraction of less than 20% ofoligomers with DP<15 and a weight fraction of less than 20% with aDP>30.
 19. (canceled)
 20. The composition of claim 1, wherein thefraction of N-acetylated units (F_(A)) of said chitosan oligomers isless than 0.1.
 21. The composition of claim 1, wherein the fraction ofN-acetylated units (F_(A)) of said chitosan oligomers is less than 0.01.